Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family - PubMed (original) (raw)
Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family
H Li et al. Proc Natl Acad Sci U S A. 2000.
Abstract
IL-17 is a T cell-derived cytokine that may play an important role in the initiation or maintenance of the proinflammatory response. Whereas expression of IL-17 is restricted to activated T cells, the IL-17 receptor is found to be widely expressed, a finding consistent with the pleiotropic activities of IL-17. We have cloned and expressed two novel human cytokines, IL-17B and IL-17C, that are related to IL-17 ( approximately 27% amino acid identity). IL-17B mRNA is expressed in adult pancreas, small intestine, and stomach, whereas IL-17C mRNA is not detected by RNA blot hybridization of several adult tissues. No expression of IL-17B or IL-17C mRNA is found in activated T cells. In a survey of cytokine induction, IL-17B and IL-17C stimulate the release of tumor necrosis factor alpha and IL-1beta from the monocytic cell line, THP-1, whereas IL-17 has only a weak effect in this system. No induction of IL-1alpha, IL-6, IFN-gamma, or granulocyte colony-stimulating factor is found in THP-1 cells. Fluorescence-activated cell sorter analysis shows that IL-17B and IL-17C bind to THP-1 cells. Conversely, IL-17B and IL-17C are not active in an IL-17 assay or the stimulation of IL-6 release from human fibroblasts and do not bind to the human IL-17 receptor extracellular domain. These data show that there is a family of IL-17-related cytokines differing in patterns of expression and proinflammatory responses that may be transduced through a cognate set of cell surface receptors.
Figures
Figure 1
Alignment of the protein sequences of IL-17, IL-17B, and IL-17C. The putative signal sequences are underlined, potential N-linked glycosylation sites are double-underlined, and conserved tryptophan and cysteine residues are marked with asterisks. IL-17, IL-17B, and C share 26–28% amino acid identity with each other.
Figure 2
RNA blot analysis of IL-17B and reverse transcription–PCR (RT-PCR) analysis. (A) Northern blot of mRNA from human tissues hybridized to a human IL-17B-specific radiolabeled probe. RNA size markers are shown on the right. A rehybridization of the same blot with a human β-actin cDNA probe is shown at the bottom. (B) Total RNA from unactivated (lanes 1, 3, 4, 6, 7, 9, and 10) and activated (lanes 2, 5, 8, and 11) CD4+ T cells was RT-PCR-amplified with primers to the 5′ and 3′ ends of human IL-17 (lanes 1–3), IL-17B (lanes 4–6), or IL-17C (lanes 7–9) coding regions. As a positive control, DNA fragments containing either IL-17 (lane 3) or IL-17B (lane 6) or IL-17C (lane 9) coding regions were diluted (1:10,000) into the cDNAs made from untreated total RNA. cDNA samples also were amplified by using human β-actin primers (lanes 10 and 11).
Figure 3
Biological activities of IL-17, IL-17B, and IL-17C. (A) Human foreskin fibroblast (HFF) cells were cultured with IL-17.Fc, IL-17B.Fc, IL-17C.Fc, or the control Fc fusion protein, Flt4.Fc (50 nM each), for 18 hr, and the conditioned media were assayed for IL-6 as described in Materials and Methods. Human leukemic cell line THP-1 was treated with the same cytokines (50 nM each) as above under the same conditions, and the supernatants were assayed for the level of TNF-α (B) or IL-1β (C). Results are expressed as the mean ± SE of triplicate determinations from one representative experiment.
Figure 4
Time course and concentration dependence of IL-17B- and IL-17C-activated TNF-α release from THP-1 cells. (A) THP-1 cells were incubated with 50 nM IL-17B.Fc or IL-17C.Fc for 0.5–48 hr, the conditioned media were harvested, and the TNF-α concentration was quantitated as described in Materials and Methods. (B) THP-1 cells were treated with IL-17B.Fc and IL-17C.Fc at a concentration range from 0 to 150 nM for 18 hr, and the TNF-α release was determined.
Figure 5
Immunoprecipitation of IL-17R ECD with IL-17, IL-17B, and IL-17C. His-tagged IL-17 receptor ECD was expressed in 293 cells and metabolically labeled with 35S as described in Materials and Methods. The supernatant was collected. To examine the expression of the IL-17R ECD, Ni-NTA beads were used to affinity-precipitate the His-tagged IL-17R ECD in the supernatant (lane 1). (A) IL-17, IL-17B.Fc, IL-17C.Fc, or the control Fc fusion protein was incubated with the supernatant, and protein A-agarose beads were added to precipitate the Fc fusion proteins. For IL-17 immunoprecipitation reaction, anti-IL-17 antibodies were included. (B) For the competitive binding experiment, immunoprecipitation of IL-17R ECD by IL-17 was performed in the presence of a 5-fold excess of IL-17B.His and control His-tagged proteins. Precipitates in both A and B were analyzed by electrophoresis on NuPAGE (4–12% Bis-Tris) gels. Molecular mass markers are indicated at the left of A and B.
Figure 6
Fluorescence-activated cell sorter analysis of the binding of IL-17B.Fc and IL-17C.Fc to THP-1 cells. THP-1 cells were incubated with IL-17B.Fc (A) or IL-17C.Fc (B) or the control Fc fusion protein, Flt4.Fc, in PBS (5% horse serum) and followed by the addition of FITC-conjugated anti-Fc secondary antibodies.
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